Journal of Natural Products Vol. SO. No. 5 , pp. 989-998, Sep-Ort 1987
989
ISOLATION OF CYTOCHALASINS A AND B FROM ASCOCHYTA HETEROMORPHA
'
R. CAPASSO,+A. EVIDENTE,G. RANDAZZO, A. RITIENI,
Dipartimento di Chimica Organira e Biologira, Uniwrsita di Napoli, via Mezzocannone 16. 80134 Napoli, Ita12 A. BOTTALICO,M. VURRO,and A. LOGRIECO
Istituto Tossinee Mirotossine h Parassiti Vegetali del C N R and Dipartimento di Patologia Vegetaledell'Universita, via Amenahla 197/F, 70126, Bari, Italy A severe foliar blight of oleander (Nerium olean& L.) caused by Asrorhyta heteromotpha (Sch. et Sacc.) Curzi, characterized by extensive brown-leaf zonations, was observed in a nursery near Bari during April of 1985. Investigation on the toxic metabolites responsible for the foliar symptoms led to the purification in large amount of cytochalasins B (1-3) and a smaller amount of A (1,2), both known for their biological activity (4). It is reported that Helminthosporium dematioideum and Phoma spp. produce cytochalasins A and B (5), and Hormiscium spp. producing only cytochalasin B (5). Moreover, it has been reported that the only phytopathogenic fungus, producing cytochalasins, specifically H and J, is Phomopsispaspalli (6). The role ofthe cytochalasins A and B in the observed oleander leafblight is under investigation. However, it appears that their importance is very limited because most of the lipophilic phytotoxic activity remains in the organic chromatographic fractions after their purification. Furthermore, a very strong phytotoxic activity was found still in the same culture filtrate after exhaustive CH,CI, extraction. EXPERIMENTAL FUNGUSSPECIES.-A. heteromotpha was isolated from brown spots of oleander leaves and was maintained on slants of potato-dextrose-agar in the collection of Istituto Tossine e Micotossine da Parassiti Vegetali del CNR, Bari, Italy. PRODUCTION, ISOLATION, AND CHARACTERIZATION OF CYTOCHALASINS A AND B .-Single spore cultures of A . heteromotpha, freshly reisolated from infected oleander plants, were used. Fungus was cultured in Roux flasks containing a semisynthetic liquid medium (7), incubated at 25' for 2 1 days. The culture filtrates (9 liters, pH=7) were lyophilized, resuspended in distilled H,O, and extracted with CH,CI,. The organic extracts were combined, dried, and evaporated under reduced pressure. The residue (1.0 g) was chromatographed on SiO, column, which was eluted with a discontinuous EtOAc-n-hexane gradient starting with a 60:40 mixture. The fractions (EtOAc-n-hexane, 80:20) showing phytotoxic activity gave a residue of 125 mg. This material, after crystallization from EtOAcIn-hexane, yielded a white crystalline compound (90 mg, 10 mg for each liter ofculture broth) that showed no phytotoxicity when assayed on tomato cuttings. The toxic activity remained in the mother liquors; its nature is under further investigation. The mp (218-220O)of the crystalline compound, its elemental analysis (found: C, 69.95; H , 7.70; N , 2.80%; M, 479, C,9H3,N0,XH,0), and its uv, ir, ms, 'H-and ' k - n m r spectra were identical to those ofan authentic sample ofcytochalasin B and were consistent with the data reported in the literature (1-3). Reaction of cytochalasin B dissolved in pyridine with Ac,O under the usual conditions gave a derivative which showed physical and spectral data consistent with those of diacetykytochalasin B (1,2). The mother liquors of the cytochalasin B crystallization yielded, by evaporation under reduced pressure, an oily mixture containing cytochalasin A as the main component, together with the other compounds responsible for the phytotoxicity of the mentioned fraction (35.4 mg). The mixture was further purified on a SiO, column (eluent: CHCL,-iPrOH, 95:5) giving a homogeneous compound (25 mg). Crystallization from EtOAcln-hexane yielded a white solid (16 mg, 1.8 mg/liter). The mp (181-183"), the ir, ms, uv, and 'H-nmr data were identical to those of an authentic sample of cytochalasin A and were consistent with data reported in the literature (1,2). Cytochalasin A, compared to B, showed the lack ofthe signal of H-20 in the 'H-nmr spectrum and the downfield shift to 203 ppm (A= 132.6) ofC-20 in the "Cnmr spectrum. The oxidation of cytochalasin B by MnO, in CH,CI, afforded a product identical in all respects to cytochalasin A. Full details on the isolation of the compounds are available upon request to the senior author.
'Present address: Istituto di Chimica Biologica Facolta di Medicina e Chirurgia Universita degli Studi di Cagliari, Italy.
Journal of Natural Products
990
[Vol. 50, No. 5
ACKNOWLEDGEMENTS
This investigation was supported by the Italian National Research Council (CNR), a special ad hoc program "Chimica Fine e Secondatia," subproject Ca. LITERATURE CITED 1. 2. 3.
4. 5.
6. 7.
D.C. Aldridge, J.J. Armstrong, R.N. Speake, and W.B. Turner,J. Chem. SK. C, 1667 (1967). W. Rothweiler and Ch. Tamm, Heh. Chim. Adu, 53 (4) 696 (1970). W . G d , J.L. Robert, J.C. Vederas, C. Tamm, P.H. Salomon, I. Miura, and K. Nakanishi, Hdu. Chim. Adu, 57 (6), 1801 (1967). S. Natori, in: "Mycotoxins in Human and Animal Health," Pathotoxic Pub. Inc., Park Forest-South Illinois, 1977, pp. 559-581. M. Binder and C. Tamm, Angew. Chem. Internat. Edit., 12,370 (1973). S.A. Patwardhan, R.C. Pandey, and Sukh Dey, Phytwhemistry, 13, 1985 (1974). A. Nachmias, I. Barash, Z. Solel, and G.A. Strobel, Physiof. PI. Pathof.,10,147 (1977).
Raeiwd 29 October I986
ESTRAGOLE: AN ACUTE TOXIC PRINCIPLE FROM THE VOLATILE OIL OF THE LEAVES OF CLAUSENA ANISATA
ADEWOLEL. OKUNADE+ Department of Chemistry, and JULIUS I. OLAIFA
Depurtment of Plant Science, Uniwrsity of lfe, Ife-lf, Nigeria The toxicity of the volatile oil of the leaves of C/uusenuunisataHook. f. (Rutaceae) to variegated grasshopper Zaorerrrs uuriegutus L. is reported. The plant species has a strong, aromatic odor compared by some to aniseed and by others to garlic (1). Its uses in some parts of Africa and the Phillipines had earlier been reviewed (1). One of the many uses is the burning of the dried plant to repel mosquitoes. Novak also demonstrated that the volatiles from the leaves ofthe plant had repellent activities against a tick, Ixodes ricims (2). Steam distillation of the fresh leaves yielded a strong, sweet-smelling, brownish-yellow oil. Acute toxicity tests on the oil showed that it was toxic to the third nymphal instar of the grasshopper. Chromatography led to the isolation of estragole (the major component of the oil) which was 1!h times more toxic than the crude oil. This represents the first report of the biological activity of this compound. Furthermore, its isolation as the major compound from the volatile oil of the leaves of this plant contradicts the earlier report of anethole as the major constituent (1). EXPERIMENTAL
GENERAL EXPERIMENTAL PROCEDURES.-The ir and UV Spectra were obtained on b e UniCam Sp3-300 and $8-400 spectrometers, respectively. The 'H-nmr spectra were obtained on a Varian FT 80 A spectrometer (80 MHz) and the ',C-nrnr spectra were recorded on a Varian FT 80 A spectrometer (20 MHz). CDCI, was used as solvent with TMS as internal standard. EXTRACTION AND ISOLATIONOF ESTRAGOLE.4+366 (hexane-EtOAc, 10:1). The fastest-moving component (Rf 0.78) was collected and extracted from the silica with CHCI,. Concentration of the CHCI, extract left a colorless oil (70% of the total oil) that was identified as estragole [ l-methoxy-4-(2'-propenyl)benzene) by comparison of its spectral data (uv, ir, 'H nmr) with published data (3,4) and the ',C-nmr data: 158.1 C-I, 137.9 C-2', 132.1 C-4, 129.5 C-3 and 5, 115.4C-2and6, 113.9C-3', 55.2-OCH3, 39.4C-1'.
